TABSAT

TABSAT - Targeted Amplicon Bisulfite Sequencing Analysis Tool - is a tool for analyzing targeted bisulfite sequencing data generated on an Ion Torrent PGM / Illumina MiSeq. It performs

• Quality Assessment
• Alignment using Bismark
• Result aggregation into a table
• Visualization as lollipop plots

Available as

• Fully configured Docker image Dockerfile - see usage information below.
• Source code

Collaboration

Please contact us if you need help running your analyses. Also we have developed an extended version for our collaborators with the following additional features:

• Interactive web-based visualization
• Download FASTA of target regions
• Strand specific CpGs
• Automatic mapping of primers
• Restriction enzyme positions
• Start using web frontend
• Pattern visualization and analysis

Publication

TABSAT is published:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160227

Example usage

${TABSAT} -l NONDIR -g hg19 -q 20 -m 10 -p 0.8 -r 0 -t target.csv -a tmap -o output_dir input.fastq

-t Targetlist in CSV format example [mandatory] - Strand can be "+", "-", "+/-"
-e Sequencing library - SE/PE (PE reads must be called *_1.fastq, *_2.fastq)
-g Genome (hg19, mm10)
-l Library mode of bisulfite experiment
-a [optional] Specify the aligner that should be used
-m [optional] This parameter is used for filtering reads that are shorter than the given threshold.
-q [optional] Bases that are below the given threshold are removed from the 3’ end of the reads (read trimming)
-p [optional] Percent of target covered by a read for pattern creation. This value specifies the percent of the target that needs to be covered by a read to include it for pattern analysis.
-r: [optional] Minimum number of mapped reads that need to be present at each CpG site.
-s: [optional] Sorted list of samples that is used to specify the order in the lollipop plots.
-o Output directory
-d Directory of inputfiles (absolute path); if not specified, the input files are added at the end [optional]

Examples

Test with input file directory

tabsat -l NONDIR -g hg19 -t target.csv -d test_input_dir -a tmap -o test_output_dir

Test with separate input files

tabsat -l NONDIR -g hg19 -t target.csv -o test_output_files xy.fastq abs.fastq

Test data

Test data is available here

Installation

• Check out the project (git clone)
• Download the reference genome
• Human
  • Broad: ftp://[email protected]/bundle/2.8/hg19/ucsc.hg19.fasta.gz
  • ENSEMBL: ftp://ftp.ensembl.org/pub/release-75/fasta/homo_sapiens/dna/Homo_sapiens.GRCh37.75.dna.primary_assembly.fa.gz
  • NCBI: ftp://ftp.ncbi.nlm.nih.gov/genbank/genomes/Eukaryotes/vertebrates_mammals/Homo_sapiens/GRCh37.p13/seqs_for_alignment_pipelines/GCA_000001405.14_GRCh37.p13_no_alt_analysis_set.fna.gz
• Mouse
  • USCS: http://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips/mm10.2bit
• Put the reference genome file into the correct folder
  • Human
    tabsat/reference/human/hg19/hg19.fasta
  • Mouse
    tabsat/reference/mouse/mm10/mm10.fasta
• Prepare the reference genome

$ tabsat/reference/prepareReference.sh

• Prepare the CpG file

apt-get install p7zip-full
7za e tabsat/tools/ait/all_cpgs_only_pos_hg19.7z
7za e tabsat/tools/ait/all_cpgs_only_pos_mm10.7z

• Install Perl modules
  • Cairo.pm
  • Switch.pm
• Run 'install' script in tabsat folder (installs SAMtools, Bedtools) ./install

Run example

Command line

• After installation go to tabsat/tools/zz_test
• Execute

./test_tabsat.sh

• Inspect output at tabsat/tabsat_test_output

Docker

• Build the docker file
  docker build -t tabsat:v1 .

• Run it
  docker run -t --name tabsat -d tabsat:v1

• Connect to docker
  docker exec -ti tabsat /bin/bash

• Stop container
  docker stop tabsat

• Remove container
  docker rm tabsat

• Remove image
  docker rmi tabsat:v1